There is also massive recruitment of SEC31 to endoplasmic-reticulum exit sites. Furthermore, absence of PHB decreases the levels of the Golgi-localised KDELR, thus preventing KDELR-dependent activation of Golgi-Src and inhibiting Golgi-to-plasma-membrane transport of VSVG. We propose a model whereby in analogy to previous findings , PHB can act as a signalling scaffold protein to assist in KDELR-dependent Src activation.The KDEL receptor (KDELR) is a seven-transmembrane-domain protein involved in retrograde transport of protein chaperones from the Golgi complex to the endoplasmic reticulum. Our recent findings have shown that the Golgi-localised KDELR acts as a functional G-protein-coupled receptor by binding to and activating Gs and Gq. These G proteins induce activation of PKA and Src and regulate retrograde and anterograde Golgi trafficking. Here we used an integrated coimmunoprecipitation and mass spectrometry approach to identify prohibitin-1 (PHB) as a KDELR interactor. PHB is a multifunctional protein that is involved in signal transduction, cell-cycle control, and stabilisation of mitochondrial proteins. We provide evidence that depletion of PHB induces intense membrane-trafficking activity at the ER–Golgi interface, as revealed by formation of GM130-positive Golgi tubules, and recruitment of p115,  Intracellular organelles maintain their homeostasis through the continuous exchange of proteins and lipids, which tend to intermix during membrane trafficking. The transport of cargoes from the endoplasmic reticulum (ER) to the Golgi complex involves temporary mislocalisation of ER-resident proteins into post-ER compartments. By virtue of their KDEL sequence, these ER proteins can bind to the KDEL receptor (KDELR) and are thence shuttled back to the ER. To date, three different genes have been identified in human that encode the closely related KDELRs: KDELR1, KDELR2, and KDELR3 .α-synuclein, and the pathological huntingtin [The KDELR is an integral membrane protein with seven transmembrane domains . The N-tntingtin . Recentlntingtin , 11. Herntingtin , 11. Thentingtin .In the present study, while looking for novel KDELR interactors that might participate in the signal transduction of the KDELR, we identified prohibitin-1 (PHB). PHB is a member of an evolutionarily conserved family of proteins that includes PHB-2, stomatin, erlins, flotillins, and the bacterial protein HflK. These proteins share the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain, which is also known as the PHB domain . PHB-domPHB is a 30-kDa protein with a single membrane spanning domain mainly localised in the inner mitochondrial membrane, where it functions as a chaperone and controls proteostasis of mitochondrial proteins , 18. PHBIn the present study, we have identified a KDELR–PHB complex and explored the functional role of PHB along the KDELR signalling pathway, as well as in its coordination of membrane trafficking. We provided evidence that PHB is required for KDELR-dependent SFKs activation, transport of cargo from the Golgi complex to the plasma membrane, and membrane trafficking between the Golgi complex and the ER.To elucidate the molecular events triggered by the KDELR in the regulation of membrane trafficking, we used a coimmunoprecipitation approach. The best antibody available against the endogenous KDELR recognizes an epitope sequence at the KDELR C-tail. As this region is believed to be involved in binding with KDELR interactors, to prevent any bias in our immunoprecipitation, we used a transfected epitope-tagged version of the KDELR . Furtherhttp://dx.doi.org/10.1155/2015/319454). Also, the structure of the Golgi complex was not affected in the HeLa-myc cells, as assessed by GM130, mannosidase II and TGN46 staining . The membrane-traffic efficiency was investigated using the temperature-sensitive mutant vesicular stomatitis virus G glycoprotein (VSVG). This mutant VSVG is synchronisable according to temperature, and it has been widely used to assess protein folding and the efficiency of the secretory pathway [Initially, we characterised this cell line to determine whether this stable transfection of the myc-tagged KDELR affects distribution of the endogenous KDELR, Golgi complex organisation, or membrane transport. Here, the intracellular distribution of the stably transfected myc-tagged KDELR was similar to that of endogenous KDELR, and their expression levels were also comparable . These proteins were absent in the immunoprecipitate obtained from the wild-type control HeLa cell lines. These data indicated that the chosen cell model and experimental conditions are optimal to reveal KDELR interactors.To determine whether the immunoprecipitation conditions were appropriate for coimmunoprecipitation of the KDELR with its interacting partners, we searched for proteins that should interact with the KDELR on the bases of previous studies, or that are components of the retrograde transport machinery. The HeLa-myc and control HeLa cells were lysed, and the proteins were immunoprecipitated using agarose-conjugated anti-c-myc antibodies. The immunoprecipitated proteins were analysed by Western blotting, which revealed the Gs and Gq subunits of the heterotrimeric G proteins , 25, andTo identify KDELR interactors, we carried out preparative coimmunoprecipitation of the KDELR from the control and HeLa-myc cells. The immunoprecipitated proteins were separated by two-dimensional gel electrophoresis, and then the gels were silver stained. Differentially immunoprecipitated proteins were excised, subjected to tryptic digestion, and analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The peptide masses obtained were matched to peptide mass databases using the ProFound and MASCOT software. Peptide matching and protein searches were performed by submitting the peptide mass lists to database searches on NCBInr and/or SWISS PROT, using the MASCOT and ProFound search engines.This analysis identified PHB as a potential KDELR interactor. In view of our interest in the signalling functions of the KDELR, PHB was further investigated.To confirm that PHB is part of the KDELR interactome, HeLa-myc cells stably expressing the KDELR-myc chimera were subjected to coimmunoprecipitation using an agarose-conjugated anti-c-myc antibody, followed by Western blotting with an anti-PHB antibody. This approach confirmed coprecipitation of PHB with the KDELR . To exclμg/mL BFA for 5 min and then subjected to coimmunoprecipitation using an agarose-conjugated anti-c-myc antibody, which was followed by Western blotting with an anti-PHB antibody. After this 5 min of BFA treatment the KDELR lost its interaction with PHB  . The funwith PHB . These d cis-Golgi marker GM130 and KDELR-GFP, indicating that the Golgi membranes can be targeted by PHB  is a protein complex that coats newly forming membrane carriers , and participates in intra-Golgi transport and retrograde Golgi-to-ER transport. β-COP is a component of the COPI complex, and it has been used to monitor the activity and distribution of COPI. Golgi BFA-resistant guanine nucleotide exchange factor 1 GBF1 is an activator of the small GTPase ARF and is also a part of the COPI complex. COPII is a protein complex that coats newly forming membrane carriers (vesicles and tubules) that exit the ER. SEC31 is a component of COPII, and it has been used to monitor the activity and distribution of COPII.To gain insight into the role of PHB at the Golgi complex, we carried out morphological analysis of the secretory pathway in cells under RNA interference for PHB. HeLa-myc cells were treated with PHB small-interfering (si)RNA, and PHB expression was analysed at various times. PHB was down-regulated by 70% already after 48 h of this RNA interference, and reached about 90% knock-down after 96 h . This laβ-COP immunofluorescence at the Golgi as well as on peripheral dots  was also increased  cells . Specifi Figures . GBF1 sh Figures . Similar Figures . FinallyAltogether, these findings suggest that removal of PHB stimulates membrane trafficking between the Golgi and the ER.Our group reported that KDELR stimulation triggers a signalling cascade that activates SFKs and controls membrane trafficking . Here, wOur previous study showed that a transport pulse of VSVG activates the KDELR, and in turn, the SFKs . Thus, PAccording to our published model, cargo proteins are transported from the ER to the Golgi complex together with KDEL proteins. These KDEL proteins can then bind to the KDELR, and activate the signalling cascade and the SFKs . Thus, aTo determine whether the SFKs activation and inhibition observed in PHB-depleted cells is caused by a transport problem, we monitored VSVG transport from the ER to the Golgi complex. Indeed, VSVG was efficiently transported to the Golgi complex in PHB-interfered HeLa-myc cells .SFKs inhibition results in the accumulation of VSVG in the Golgi complex, which prevents its arrival at the plasma membrane . Here, wHeLa-Myc cells with PHB knocked down were infected with VSV, and transport of VSVG was monitored 30 min and 100 min after the release of the traffic block. At 30 min, in the control (mock interfered) and PHB-interfered cells, VSVG was mainly localised in the Golgi complex. At 100 min, VSVG had reached the plasma membrane in the controls, whereas it remained in the Golgi complex in PHB-interfered cells . This inER-resident proteins bearing the KDEL motif can reach post-ER compartments during membrane trafficking –32. The To understand how PHB knock-down might hinder KDELR–SFKs activation, we investigated the distribution of the KDELR in PHB-interfered HeLa cells. Immunofluorescence analysis of the endogenous KDELR showed major amounts in the Golgi complex and lesser amounts in the ER. PHB-depleted cells showed a striking reduction in the KDELR in the Golgi complex Figures . WesternThese reduced levels of endogenous KDELR in the Golgi complex of PHB-depleted HeLa cells might be caused by an imbalance in the cycling of the KDELR between the ER and the Golgi complex. We investigated this possibility using an artificial chimeric protein constituted by the luminal domain of VSVG linked to the N-terminus of the KDELR (VSVG–KDELR). Previous studies demonstrated that VSVG–KDELR accumulates in the Golgi complex when cells are incubated at 32°C, while at 40°C, VSVG–KDELR accumulates in the ER. Shifting the temperature of incubation of the cells from 32°C to 40°C, it is possible to follow retrograde transport of the KDELR from the Golgi to the ER. PHB-interfered COS-7 cells were transfected with the VSVG–KDELR chimera and incubated overnight at 32°C. The day after, the cells were incubated at 40°C for 2 h, and the localisation of the chimera was analysed by confocal microscopy. VSVG–KDELR accumulated in the Golgi complex of controls and PHB-interfered cells upon incubation at 32°C .This all indicates that PHB depletion affects KDELR homeostasis by accelerating its retrograde movement from the Golgi to the ER. The reduced levels of KDELR at the Golgi complex can contribute to decreased KDELR-dependent activation of SFKs in PHB knock-down cells. cis-Golgi, which results in activation of retrograde membrane transport [The KDELR belongs to the PQ-loop protein family , which iransport , while Gransport .In the present study, to uncover the molecular players involved in the KDELR signal-transduction machinery, we searched for interactors of the KDELR using a coimmunoprecipitation and mass spectrometry approach. We identified PHB among the proteins that coimmunoprecipitated with the KDELR from HeLa cells. The KDELR–PHB complex was validated by modulation of their interactions upon BFA treatment. BFA inhibits the guanine-nucleotide exchange factors on ADP-ribosylation factor (ARF-GEFs), thus resulting in inactivation of ARF and loss of the KDELR–PHB interaction. This suggests that the KDELR–PHB complex relies on a functional ARF1.To date, intracellular PHB has been reported in the mitochondria and nucleus and at the plasma membrane but not in the ER or the Golgi complex, while the KDELR cycles between the ER and the Golgi , 29. In  Saccharomyces Genome Database (http://www.yeastgenome.org/).However, to better support this novel relationship between the KDELR and PHB, we carried out a database analysis, with a search for any PHB interactors that have a well-established localisation to or function in the ER or the Golgi complex. Remarkably, PHB interacts genetically with the following yeast proteins: ARV1, an integral membrane protein that cycles between the ER and the Golgi complex and is involved in sphingolipid transport ; ERG5, a cis-Golgi and a component of the alpha-1,6 mannosyltransferase complex [2+/Mn2+ ions into the Golgi complex [ Saccharomyces Genome Database (http://www.yeastgenome.org/). Finally, the α subunit of the human COPI complex has been identified in a complex with PHB .Furthermore, the yeast PHB protein has been detected in complexes with: ERP1, which is a member of the p24 family proteins and a component of the KDELR functional machinery ; MNN9, M complex ; and PMR complex . These dThese numerous relationships among PHB and components of the ER and Golgi include proteins that are strictly related to the KDELR , and they strongly support the presence of PHB in these organelles, and its functional relationship with the KDELR.PHB has an important role in the signalling pathway triggered by the RAS oncogene . Indeed, cis-Golgi location and to allow activation of the SFKs.Here, we have reported that the knock-down of PHB mislocalises the KDELR  and inhFinally, in agreement with our previous data that showed that inhibition of KDELR-SFKs signalling impairs the transport of VSVG from the Golgi complex to the plasma membrane , the preβ-COP monoclonal ; rabbit anti-lysozyme polyclonal (Chemicon); rabbit anti-p-SFKs polyclonal (p-Tyr418) ; rabbit anti-SFKs polyclonal (Santa Cruz Biotechnology); mouse anti-GM130 monoclonal and mouse anti-GBF1 monoclonal ; the mouse P5D4 anti-VSVG monoclonal (Sigma Aldrich); and rabbit VSVG luminal domain polyclonal (A. De Matteis), Rabbit p115 [The following antibodies were used: rabbit anti-c-myc polyclonal (Santa Cruz); rabbit anti-PHB polyclonal ; mouse anti-bit p115  and SEC3Cell Handling. Human HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum, 2 mM L-glutamine, 100 μg/mL streptomycin sulphate, and 100 units/mL penicillin G , at 37°C in a humidified 5% CO2/air atmosphere. HeLa cells stably transfected with the human myc-tagged KDELR (HeLa-myc cells) were kindly provided by W. Hsu.Transfections. The cells were transfected with Fugene 6  according to the manufacturer instructions. RNA Interference. The cells were transfected with Lipofectamine (Thermo Fisher Scientific Inc.), according to manufacturer instructions. PHB siRNA LQ-010530-00 . VSV Infection. The cells were infected with VSV as previously described . Transport Pulse Protocols. The VSVG transport pulses were as previously described . Cycloheximide  was added at 50 μg/mL at the temperature shift.3VO4, 5 mM PMSF, 5 μg/mL each leupeptin, aprotinin, pepstatin), at 4°C. The cell lysates were centrifuged at 15,000 ×g for 5 min at 4°C, to pellet and remove the nuclei. The postnuclear supernatant was immediately processed for SDS-PAGE and Western blotting. Of note, this separation of the postnuclear supernatant from the nuclei can be crucial for the detection of tyrosine phosphorylation.Following the transport protocol, the cells were washed three times with ice-cold PBS and harvested immediately in lysis buffer  were cooled on ice, washed with ice-cold 0.9% NaCl (3 times) , scraped, and lysed with 1.8 mL ice-cold lysis buffer  and a cocktail of protease inhibitors (Roche)). Subsequently, the total lysates were passed through a syringe needle (15 times) and incubated on a rotating wheel for 1.5 h at 4°C. The lysates were then cleared by centrifugation at 14,000 rpm for 15 min. The supernatants (4–6 mg protein) were incubated overnight with agarose beads coupled to rabbit polyclonal c-myc antibody (50 μL/mg) (Santa Cruz Biotechnology). The settled beads were extensively washed with lysis buffer, and the bound protein was eluted 10 times with 0.1 M ammonium hydroxide, pH 11. The eluted samples were dialysed against 500 mM ammonium hydroxide, pH 11, overnight at 4°C, and then concentrated by lyophilisation.To prepare total cell lysates, cells , and 1% IPG buffer (Amersham)). Proteins were separated by isoelectric focusing using an IPGphor apparatus (Amersham). In-gel rehydration was carried out on immobilised 13-cm IPG strips with a broad pI range: pH 3–10 linear gradient (Amersham). The optimised isoelectric focusing conditions were 20°C, 50 For the second dimension, the IPG strips were equilibrated for 15 min in 6 M urea, 50 mM Tris-HCl, pH 8.8, 30% glycerol, 2% SDS, 1% dithiothreitol, and for 15 min in 6 M urea, 50 mM Tris-HCl, pH 8.8, 30% glycerol, 2% SDS, 100 mM iodoacetamide. Proteins were separated using 12.5% SDS-PAGE, following standard protocols, and revealed with mass-spectrometry compatible silver staining.2O/0.1% trifluoroacetic acid at 30°C, for 15 min. The resulting tryptic peptides were extracted, desalted with ZipTip C18 columns , directly eluted, and crystallised in a saturated solution of α-cyano-4-hydroxy-cinnamic acid in 50% (v/v) acetonitrile/H2O.Protein bands were excised from the SDS-PAGE and placed in 0.5 mL microcentrifuge tubes . After washing, the cysteines were reduced and alkylated with iodoacetamide . The samThe samples were then spotted onto stainless steel MALDI sample target plates, and the peptide mass spectra were obtained by MALDI TOF MS , with a nitrogen laser and an emission wavelength of 337 nm. The mass spectra were acquired in positive-ion reflectron mode with delayed extraction and 20 kV acceleration voltage. External calibration was performed for each measurement, using a mixture of seven standard peptides . All of the mass spectra were acquired using a minimum number of 250 laser shots. The spectra were internally calibrated with trypsin autolysis products. Peptide matching and protein searches were performed through submission of the peptide mass lists to database searches (NCBInr and/or SWISS PROT), using the Mascot and ProFound search engines.4Cl; Sigma Aldrich) in PBS for 30 min at room temperature. The cells were subsequently incubated with the specified antibodies diluted in blocking solution, for 2-3 h at room temperature, or overnight at 4°C. After incubation with the primary antibody, the cells were washed three times in PBS and incubated with a fluorescent conjugated anti-IgG secondary antibody for 1 h at room temperature. For the triple labelling (rabbit/mouse/sheep), Alexa 488-conjugated anti-rabbit or anti-mouse antibodies raised in chicken were used (rather than in goat), to avoid possible cross-reactions. The cells were finally examined under a confocal microscope . The quantification of fluorescence signals was as follows. The area of interest was delineated manually and the fluorescence intensity was quantified using the LSM510-3.2 software (Zeiss). To assess the colocalization we removed the background immunofluorescence and used the colocalization functions of the LSM510-3.2 software (Zeiss).The cells were grown to subconfluent density on glass coverslips for 24 h and then washed with PBS. Following fixing in 4% paraformaldehyde (Sigma Aldrich) for 10 min at room temperature, they were incubated in blocking solution  was similar to that of endogenous KDELR. Also, the Golgi morphology was not affected in the HeLa-myc cells, as assessed by GM130, mannosidase II and TGN46 staining.Supplementary Fig S2. shows that the transport efficiency of VSVG in HeLa-myc cells was similar to that in wild-type HeLa cells. Supplementary Fig S3. shows that the β subunit of the COPI coatomer complex coimmunoprecipitated with the KDELR from HeLa-myc cell lysate.


Stress is related to heavy alcohol use and relapse in alcoholics. Using the reinstatement model, we have shown that corticotropin-releasing factor (CRF) underlies stress-induced relapse to alcohol seeking in laboratory rodents. Little is known about how other neurotransmitters interact with CRF in these effects. Dynorphin and its receptor  are involved in stress responses and in alcohol seeking. KOR and CRF receptors (CRF R) may interact in the production of stress-related behaviors but it is not known whether this interaction is involved in reinstatement of alcohol seeking.Male Long Evans rats were trained to self-administer alcohol (12% w/v). After extinction of responding, we determined the effects of the KOR agonist, U50,488  on reinstatement of alcohol seeking, and their sensitivity to the selective KOR antagonist nor-binaltorphimine dihydrochloride (nor-BNI) (10 mg/kg) administered at different times before U50,488. We then examined the effects of nor-BNI on reinstatement induced by the stressor yohimbine (1.25 mg/kg) and on reinstatement induced by exposure to alcohol-associated cues. Finally, we determined whether CRF R1 blockade with antalarmin  attenuates alcohol seeking induced by U50,488.U50,488 reinstated alcohol seeking. Prior treatment with nor-BNI 2, but not 24 h before administration of U50,488 or yohimbine blocked reinstatement induced by these drugs. Cue-induced reinstatement was blocked by nor-BNI administered 2 h prior to testing. Finally, U50,488-induced reinstatement was blocked by antalarmin.These data further support a role for KOR in reinstatement of alcohol seeking under nonstress and stressful conditions and that KOR and CRF R interact in these effects. We will then examine the effects of nor-BNI on reinstatement induced by the pharmacological stressor yohimbine, a drug that we have shown to induce reinstatement in a CRF R1-dependent manner. Nor-BNI will be administered at two different pretreatment times prior to U50,488 or yohimbine, as its selectivity for KOR versus other opioid receptors has been shown to vary in a time-dependent manner. Then we will examine whether reinstatement of alcohol induced by cues previously associated with alcohol seeking is blocked by nor-BNI. Finally, we will determine whether blockade of CRF R1 with antalarmin attenuates alcohol seeking induced by U50,488.The experimental procedures followed the “Guide for the care and use of laboratory animals” . Food and water were freely available in the home cage at all times and the temperature was maintained at 21 ± 1°C.Long Evans rats  weighing 200–250 g at the start of the experiment were used. The rats were individually housed under a 12:12 h light-dark cycle  activated an infusion pump , while responding on the other lever (an inactive lever) was recorded, but did not activate the pump. Activation of the infusion pump resulted in the delivery of 0.19 mL of alcohol into a drinking receptacle located between the two levers and initiated a 5-sec timeout period. During the timeout, the house light was turned off, a key light over the active lever was turned on, and white noise was emitted by a speaker. The beginning of the sessions was signaled by the illumination of a houselight located at the top of the self-administration chamber; at the end of the session, the houselight was turned off. When alcohol was left in the drinking receptacle after a self-administration session, it was measured and the volume was taken into account for intake calculation.Rats were trained to self-administer alcohol as described previously  and yohimbine was obtained from Sigma-Aldrich . The doses of yohimbine and antalarmin and their pretreatment times were based on our published studies  were followed by Newman-Keuls post hoc tests.Statistical analyses were performed separately on the numbers of responses made on the previously active and inactive levers during the reinstatement tests. Data were analyzed with analysis of variances (ANOVAs) and significant interactions  as the factor. Once the extinction criterion was reached, rats received vehicle (water) or one of the doses of U50,488, 30 min before 1-h reinstatement test sessions conducted in the operant chambers, in counterbalanced order with at least 2 days between each test. On the days between the drug tests, rats were injected i.p. with water and received drug-free extinction sessions.n = 7–8 per group) were trained to self-administer alcohol and received extinction sessions. In this and in the following experiments, rats were assigned to matched groups based on alcohol intake and extinction responding. A mixed design was used with the between factor of nor-BNI pretreatment condition  and within factor of U50,488 pretreatment condition . One group of animals received injections of nor-BNI vehicle or nor-BNI  2 h before the U50,488 vehicle or U50,488 injections. The other group received nor-BNI 24 h prior to U50,488 vehicle or U50,488 injections. Thirty minutes after the U50,488 vehicle or U50,488 injections, rats were placed in the operant chambers for the 1-h reinstatement test session. In order to minimize the use of animals in Experiments 2 and 3, we did not include a 24 h nor-BNI vehicle condition; we have found that baseline extinction responding is extremely stable from day to day once the extinction criterion is reached.Twenty-three rats (n = 12 per group), trained to self-administer alcohol and their responding was extinguished as above. A mixed design was used with the between factor of nor-BNI pretreatment condition  and within factor of Yohimbine condition . During testing, each rat was injected with the nor-BNI vehicle 2 h before, or nor-BNI , 2 or 24 h prior to injections of the yohimbine vehicle (water) or yohimbine. Forty-five minutes after the vehicle or yohimbine injections, rats were placed in the operant chambers for the 1-h reinstatement test session.The effects of nor-BNI on yohimbine-induced reinstatement was assessed in 36 rats (n = 11 per group), trained to self-administer alcohol and their responding extinguished as above, were used to determine the effects of nor-BNI on reinstatement induced by the light/tone cue previously associated with alcohol self-administration. A mixed design was used with the between factor of nor-BNI pretreatment condition  and within factor of Cue condition . During testing, each rat was injected with the nor-BNI vehicle or nor-BNI  2 h prior to 1-h test sessions. Rats were placed in the operant chambers and received one non-contingent presentation of the tone-light cue. Subsequent presses on the active lever resulted in cue presentation on a FR1 schedule during the remainder of the session. Extinction responding without cue presentation the previous day was used as the baseline. A 24 h nor-BNI condition was not included as such pretreatment did not affect U50,488- and yohimbine-induced reinstatement in Experiments 2 and 3.Twenty-two rats (n = 12 per group) were trained and their responding was extinguished as above and they were used to assess the effect of antalarmin on U50,488-induced reinstatement. A mixed design was used with the between factor of U50,488 dose  and within factor of Antalarmin dose . On the test days, each rat was injected with the antalarmin vehicle  or one of the doses of antalarmin, and 30 min later with vehicle or U50,488. Thirty minutes after the vehicle or U50,488 injections, rats were placed in the operant conditioning chambers for a 1-h reinstatement test session. There were at least 2 days between drug tests, and on these days animals were injected with water, i.p. and received extinction sessions.Twenty-four rats  = 10.33, P < 0.05]. The effects of U50,488 were significantly different from vehicle at the 5 mg/kg dose [P < 0.05], but not at the 2.5 mg/kg dose (P > 0.05). There were no significant effects of U50,488 on responding on inactive lever responding  = 3.9, P < 0.05]. In animals treated with nor-BNI vehicle, U50,488 significantly increased active lever pressing compared to those administered its vehicle (P < 0.05). The U50,488-induced increase was blocked by nor-BNI when it was injected 2 h (P < 0.05), but not 24 h prior to U50,488 (P > 0.05). Inactive lever pressing was not significantly affected by U50,488 or nor-BNI  = 5.6, P < 0.05]. In animals administered the vehicle for nor-BNI, yohimbine significantly reinstated alcohol seeking (P < 0.05). This reinstatement was significantly reduced when nor-BNI was administered 2 h prior to yohimbine (P < 0.05), but not when nor-BNI was administered 24 h before. Analysis of inactive lever data showed a significant effect of Yohimbine condition, due to the fact that inactive responding was overall slightly higher in animals administered yohimbine. This effect achieved statistical significance in the 24 h nor-BNI group (P < 0.05). . Table .F = 9.03, P < 0.05]. When animals were exposed to cues previously associated with alcohol delivery they showed a significant reinstatement of active lever pressing, compared to the no cue baseline condition (P < 0.05). This reinstatement was blocked in animals given nor-BNI 2 h prior to the test, compared to those administered vehicle (P < 0.05). There were no significant effects on inactive lever responding  = 3.46, P < 0.05]. Compared to animals treated with the vehicle, those administered U50,488 showed a significant reinstatement of alcohol seeking , but not by 10 mg/kg. There were no significant effects of either of the drugs on inactive lever responding  and receptor binding studies suggest that this antagonist causes a long-lasting antagonism of KOR receptors, that lasts days or weeks, and that its selectivity for KOR plateaus at 2 h and becomes maximal at 24 h after administration  has been studied in mice (Land et al. There is also a CRF-KOR interaction in the aversive responses elicited by stress, which involves, in contrast, CRF R2 receptors. Place aversion induced by either CRF or a CRF R2 agonist was blocked by a KOR antagonist, but KOR agonist-induced place aversion was unaffected by CRF R2 blockade (Land et al. Our findings of nor-BNI blockade of yohimbine-induced reinstatement are consistent with this proposed mechanism, as yohimbine produces reinstatement through a CRF R1-dependent mechanism. Our results on antalarmin-induced blockade of U50,488-induced reinstatement, however, suggest a different mechanism, with an opposite relationship between the two peptides. These latter data suggest instead that KOR stimulation evokes the release of CRF, which in turn stimulates CRF R to induce reinstatement. Antalarmin blocks the effects of this released CRF on the CRF R, thereby inhibiting the reinstatement response. A brain region in which this interaction might occur is the amygdala, a critical part of the circuitry involved in responses to stress (Johansen et al. These data provide further support for the important role of KOR in reinstatement of alcohol seeking under nonstress and stressful conditions. They also indicate an interaction between KOR and CRF in reinstatement of alcohol seeking. Further studies are necessary to elaborate the role of KOR and CRF R in stress-induced alcohol seeking. A key experiment we intend to conduct is to examine the effect of nor-BNI on reinstatement induced by the stressor, i.c.v. CRF.


Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system, is also known to be important for brain development. Therefore, disturbances of GABA receptor (GABA-R) mediated signaling  during brain development may influence normal brain maturation and cause late-onset brain malfunctions. In this study, we examined whether the stimulation of the GABA-R signal during brain development induces late-onset adverse effects on the brain in adult male mice. To stimulate the GABA-R signal, we used either the benzodiazepine sleep-inducing drug triazolam (TZ) or the non-benzodiazepine drug zolpidem (ZP). We detected learning and memory deficits in mice treated with TZ during the juvenile period, as seen in the fear conditioning test. On the other hand, ZP administration during the juvenile period had little effect. In addition, decreased protein expression of GluR1 and GluR4, which are excitatory neurotransmitter receptors, was detected in the hippocampi of mice treated with TZ during the juvenile period. We measured mRNA expression of the immediate early genes (IEGs), which are neuronal activity markers, in the hippocampus shortly after the administration of TZ or ZP to juvenile mice. Decreased IEG expression was detected in mice with juvenile TZ administration, but not in mice with juvenile ZP administration. Our findings demonstrate that TZ administration during the juvenile period can induce irreversible learning and memory deficits in adult mice. It may need to take an extra care for the prescription of benzodiazepine sleep-inducing drugs to juveniles because it might cause learning and memory deficits. Normal brain development requires various neuronal signals must be activated at the appropriate timing and with the proper extent in the developmental brain  sleep-inducing drug triazolam  or the non-BZD drug zolpidem  on behavior during the adult stage -1-methyl-4H-1, 2, 4 -triazolo-1, 4-benzodiazepine; Sigma Aldrich Co. Steinheim, Germany), and Zolpidem -imidazopyridine-3-acetamide; Sigma Aldrich Co., Steinheim, Germany) were dissolved in 0.5% (w/v) methyl cellulose solution  and administered by gavage at the doses of 1 and 50 mg/kg. In the vehicle group, 0.5% (w/v) methyl cellulose solution (MC) was administered by gavage. Administration and experimental schedules are shown in Figure All animal experiments were conducted with permission from the Animal Ethics Committee at the National Institute of Health Sciences. Pregnant female C57BL/6NCrSlc mice at embryonic day 11 were purchased from Japan SLC Inc. . The mice were housed in plastic cages and maintained under a 12-h light/12-h dark cycle with water and chow  provided We conducted a behavioral battery test (BBT), including the open field test (OF), the light/dark transition test (LD), the elevated plus maze test (EP), the contextual/cued fear conditioning test (FZ), and the additional pre-pulse inhibition test (PPI). For the OF, LD, EP, and FZ methods details, refer to the Tanemura et al. . In the Hippocampal extracts were dissolved in Tris-buffered saline (pH 7.4) containing protease inhibitors  and phosphatase inhibitors . Equal volume of the total protein solutions were added to 2 × sample buffer solution . The amount of protein was quantified using Qubit protein assay kits . The protein samples (30 μg/well) were subjected to SDS-PAGE (7.5% polyacrylamide), and transferred to a nitrocellulose membrane. The membranes were blocked in Blocking-one  at room temperature for 90 min and incubated with primary antibodies, such as those against acetyl-tubulin , MAP2 (sc-20172), GluR1 , and GluR4 (SAB450126) overnight at room temperature. The membranes were then washed with phosphate buffered saline (pH 7.4) with 0.05% Tween-20 (PBS-T). The membranes were then incubated with peroxidase-conjugated secondary antibodies for 2 h at room temperature. After several PBS-T washes, the membranes were incubated with chemi-lumi one L  for signal production. The signal was photographed using an LAS-3000 . The intensity of each protein band was measured using the Gel Analyzer program in ImageJ, and normalized to the intensity of the acetyl-tubulin band using the following formula: (intensity of each protein band/acetyl-tubulin band intensity) × 100. We present each normalized band intensity as a percentage of its intensity in the Vehicle group. The changes in protein expression associated with TZ and ZP treatment were calculated as follows: Ratio (%) =  × 100.Actb and presented as fold change over baseline using the delta-delta CT method. Fold changes of relative gene expression levels compared to those of Vehicle animals were calculated  for 15 min at room temperature. They were then incubated with Super-Script II (Invitrogen) for 50 min at 42°C for reverse transcription. Quantitative real time PCR was performed using an ABI PRISM 7900 HT sequence detection system  using SYBR Premix Ex Taq  with initial denaturation at 95°C for 10 s followed by 45 cycles of 5 s at 95°C and 60 s at 60°C. Ct values were obtained. The primers were synthesized by FASMAC Co., Ltd., Japan. Expression of genes of interest was normalized to that of PrimersArc: forward 5′-TACCGTTAGCCCCTATGCCATC-3′, reverse 5′-TGATATTGCTGAGCCTCAACTG-3′c-Fos: forward 5′-ATGGGCTCTCCTGTCAACACAC-3′, reverse 5′-ATGGCTGTCACCGTGGGGATAAAG-3′Nr4a1: forward 5′- TTAAGAGGTGGGTCGGGTTC -3′, reverse 5′- GCAATCCTTCTCGCACACTA -3′ActB: forward 5′-GGACTCATCGTACTCCTGCTT-3′, reverse 5′-GAGATTACTGCTCTGGCTCCT-3′.t-tests. Values in graphs are expressed as the mean ± standard error of the mean (SEM).Statistical analysis was conducted using Prism 5.04 . Data was analyzed using Student's unpaired p < 0.05). In addition, in the contextual test, only the TZ-2w group had significantly lower freezing percentages . All other groups responded quickly to the tone and showed high freezing responses, while mice in the TZ-2w group had slower responses to the tone, which resulted in lower freezing responses . These results indicate that the administration of TZ during the juvenile period induces deficits in learning and memory. Exposure to either chemical did not lead to any changes in the results of the pre-pulse inhibition test  was increased and GluR1 and GluR4  were decreased in the TZ-2w group. Both GluR1 and GluR4 belong to the AMPA-type glutamate receptor family and are known to be necessary for long-term potentiation in the hippocampus  in the hippocampus shortly (8 h) after TZ and ZP administration during the juvenile period. We observed decreased mRNA expression of IEGs in the TZ-2w group, but not in the ZP-2w group  and several biochemical assays. We stimulated the GABA-R signal of mice by the oral administration of the sleep-inducing drugs TZ (1 mg/kg body weight [B.W.]) or ZP (50 mg/kg B.W.) during juvenile  and adult stages .The results of the BBT indicate that spontaneous activity, as measured in the open field test, was significantly increased in the TZ-11w group. Reactivity to a novel environment may be changed in mice exposed to TZ during adulthood. On the other hand, deficits in learning and memory were detected by the fear conditioning test only in the TZ-2w group. The freezing response was decreased in all three stages  of the fear conditioning test. Although, the TZ-11w, ZP-11w, and ZP-2w groups had increases in freezing % as the cycles of tone and mild foot-shock were repeated, the TZ-2w group did not have an increase in freezing %, even during the later period of the test. These results may indicate that TZ-2w mice lose their ability to make short-term memories, which are needed for quick responses in this situation. In addition, TZ-2w mice had significantly lower scores in the contextual test. This may indicate deficits in spatial memory  were decreased in response to TZ treatment. IEGs are known to be induced rapidly following neuronal activation and are considered as markers of activated neurons . As expected, TZ had a greater inhibitory effect on the juvenile hippocampus than ZP. Indeed, the expression levels of all three IEGs  at prenatal period by the administration to pregnant female mice (Tanemura et al., In conclusion, our study indicates that juvenile TZ exposure may lead to learning and memory deficits. GABA-R agonists, such as BZD, are used for the treatment of both sleep disorders and anxiety disorders in both adults and children (Chevreuil et al., YF designed and performed experiments, analyzed data, and wrote the paper. KT designed the study, developed the methodology, conducted experiments, and editing the manuscript. KI helped with designed and editing the manuscript. MI helped with behavioral test and western blotting. KA, SK helped with the design of the study. MK, JK for the supervisor and design of the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli-derived ASP hypersensitivity. Sample and clinical data collection was carried out from 576 paediatric ALL patients who were treated according to protocols from the Berlin—Frankfurt—Münster Study Group. A total of 20 single nucleotide polymorphisms (SNPs) in GRIA1 and GALNT10 genes were genotyped. Patients with GRIA1 rs4958351 AA/AG genotype showed significantly reduced risk to ASP hypersensitivity compared to patients with GG genotype in the T-cell ALL subgroup (OR = 0.05 (0.01–0.26); p = 4.70E-04), while no such association was found in pre-B-cell ALL. In the medium risk group two SNPs of GRIA1 (rs2055083 and rs707176) were associated significantly with the occurrence of ASP hypersensitivity (OR = 0.21 (0.09–0.53); p = 8.48E-04 and OR = 3.02 (1.36–6.73); p = 6.76E-03, respectively). Evaluating the genders separately, however, the association of rs707176 with ASP HSRs was confined only to females. Our results suggest that genetic variants of GRIA1 might influence the risk to ASP hypersensitivity, but subgroups of patients can differ significantly in this respect.L-asparaginase (ASP) is a key element in the treatment of paediatric acute lymphoblastic leukaemia (ALL). However, hypersensitivity reactions (HSRs) to ASP are major challenges in paediatric patients. Our aim was to investigate genetic variants that may influence the risk to  Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy, comprising approximately 25–30% of the annually registered cases of cancer among children ,2. SurviEscherichia coli ASP  and an Erwinia chrysanthemi-derived ASP (Erwinia-ASP) with more or less different pharmacokinetic, pharmacodynamic and immunogenic properties [ASP enzymes mainly derived from bacteria among which three different preparations from two bacteria are currently used: native and pegylated form of operties . Hyperseoperties .E. coli-ASP is considered as first-line treatment. Clinical HSRs occur in up to 45% of paediatric ALL patients, which necessitate the discontinuation of E. coli-ASP administration and subsequent switch to PEG-ASP or Erwinia-ASP [In BFM protocols inia-ASP –12. The inia-ASP . Howeverinia-ASP . Severalinia-ASP .GRIA1  gene located at 5q33 have been found associated with ASP allergy [Recently, a genome wide association study has been carried out in an ethnically diverse population to identify germline genetic variations contributing to the risk of ASP allergy in children with ALL. In this study variants of the  allergy . This re allergy .GRIA1 gene in the susceptibility to E. coli-ASP hypersensitivity in a large Hungarian paediatric ALL population. Additionally, we also involved SNPs in GALNT10  gene, located also at 5q33, which in the original study were also found to be associated with ASP allergy in the discovery cohort, but could not be confirmed in a relatively small validation cohort.In gene association studies, due to the high number of inconclusive results, it is generally accepted that the role of a gene or a genetic variation can only be acknowledged, if it is confirmed by independent studies. In the present study our aim was to investigate the possible roles of genetic variations in the E. coli-ASP, Kidrolase  or Asparaginase medac (Kyowa-Hakko) were available as first-line ASP preparations.Samples and clinical data collection were carried out from 576 paediatric acute lymphoblastic leukaemia (ALL) patients who were treated between 1990 and 2012 in 9 Hungarian paediatric haematology centres according to four consecutive chemotherapy protocols from the Berlin—Frankfurt—Münster Study Group . These protocols in detail have been described elsewhere ,4,17,18.E. coli-ASP dosing schedules with the number of patients included are shown in The treatment according to the different risk arms focusing on E. coli-ASP containing treatment without any HSR. Therefore, we excluded further ten patients. Finally, a total of 505 patients were included in the analysis , discontinuation of ASP administration not due to hypersensitivity (n = 1), switch to a different type of ASP not due to hypersensitivity (n = 1), genotyping call rate of the sample <50% (n = 28), significant deviation from the protocol (n = 6) and use of other treatment protocols (10). The inclusion criterion for controls was the completion of at least two blocks of analysis .The National Cancer Institute Common Toxicity Criteria (CTC) system v3.0 was used to assess the grade of hypersensitivity. For analyses, we regarded a case as asparaginase hypersensitivity when signs of allergic reactions or anaphylactic reactions CTC grade 1 and above were noted which occurred during the infusion or within 4 hours, plus the clinical team decided to discontinue, not readminister the given aparaginase preparation in the later treatments. The descriptions of the HSRs were included in the medical records of the patients.Written informed consent was obtained from the study participants or from the next of kin, caretakers, or guardians on the behalf of the minors/children participants involved in the study. All the associated documents have been stored. The study was conducted according to the principles expressed in the Declaration of Helsinki and the whole study including the informed consent procedure were approved by the Hungarian Scientific and Research Ethics Committee of the Medical Research Council .GRIA1 and GALNT10 genes were searched for in online databases. The criteria of the SNP selection were the minor allele frequency ≥10% in Caucasian population, their estimated function based on dbSNP database of National Center of Biotechnology Information  or any relevant results published previously [GALNT10 the whole 3’-UTR region were covered . The genomic DNA was extracted by using the QIAamp DNA Blood Midi Kit  according to the manufacturer’s instructions. In case of low amount or low concentration of DNA  whole genome amplification was performed by using the REPLI-g Mini Kit  according to the manufacturer’s instructions.A total of 20 SNPs (10 in each gene) were genotyped by using KASPar-on-Demand prevalidated assays  on 7900HT Fast Real-Time PCR System . The genotyping call rate for all SNPs was higher than 80%.E. coli-ASP dosage during induction phase, BFM protocol and the polymorphisms in additive (11 vs. 12 vs. 22), dominant (11 vs. 12/22) and recessive (11/12 vs. 22) models were included in the analysis as categorical covariates . Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained to estimate risks for each SNP to E. coli-ASP hypersensitivity. The analyses were performed not only for the overall ALL cohort, but also for subgroups created by gender, ALL immunophenotype (pre-B vs. T-ALL), age at diagnosis (≥10 years vs. <10 years) and risk category (standard vs. medium vs. high risk).Multi-adjusted logistic regression was performed by using IBM SPSS Statistic software, version 20.0 to test for associations. Gender, ALL immunophenotype, age at diagnosis, risk group, In order to deal with multiple comparisons the Benjamini-Hochberg false discovery rate (FDR) method with type I error rate of 5% (p≤ 6.76E-03) was applied as correction ,22.Power analysis was conducted by bootstrapping using R statistical software  in the following way. First, we simulated 1 000 data replicates by sampling with replacement from the original dataset. Next, we applied logistic regression to each generated data set, and estimated power as the percentage of cases in which the null hypothesis was rejected.E. coli-ASP hypersensitivity developed. The hazard rate of the i-th patient was modeled by logistic regression:D represents the minimum number of doses at which E. coli-ASP hypersensitivity developed by each study sample ; Xi contains the value of the covariate (SNP) that might predict hazard function differences, and α and β are the appropriate regression coefficients. Analyses were performed using R statistical software.Discrete-time survival analysis was used to assess the impact of SNPs on the minimum number of doses at which 2 goodness-of-fit test using an online application (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl).The deviation from Hardy-Weinberg Equilibrium (HWE) was analysed by χGRIA1 and GALNT10 polymorphisms on the risk to E. coli-ASP hypersensitivity in paediatric ALL patients.We investigated the impact of Out of the 20 SNPs, 18 were statistically evaluated, since two of them did not meet the requirements of HWE. The genotype and minor allele frequencies (MAF) with the results of the HWE are shown in E. coli-ASP hypersensitivity and patients without HSRs to E. coli-ASP, multi-adjusted logistic regression analyses were applied on the overall cohort and on various subgroups.To examine the differences in genotype frequencies in patients with GRIA1 gene showed statistically significant associations on certain subgroups.Due to multiple testing, statistical corrections were applied. Applying FDR = 5% (p≤ 6.76E-03) significance threshold three SNPs in E. coli-ASP hypersensitivity was not replicated in our total study cohort. The allele and genotype distributions for each SNP can be found in E. coli-ASP hypersensitivity in T-cell and pre-B-cell ALL separately, in case of rs4958351 a significant difference between the subgroups was detected. In the dominant model patients with rs4958351 AA/AG genotype showed significantly reduced risk to ASP hypersensitivity compared to patients with GG genotype in the T-ALL subgroup (OR = 0.05 (0.01–0.26); p = 4.70E-04; power = 0.78); The association of rs4958351 with E. coli-ASP hypersensitivity was 37% , medium (MR) and high risk (HR) group, respectively). We found association between two polymorphisms (rs2055083 and rs707176) and the occurrence of ASP hypersensitivity in the MR group. On the one hand, in the dominant model, patients with rs2055083 AA/AG genotype had significantly lower risk to develop ASP hypersensitivity compared to patients with GG genotype (OR = 0.21 (0.09–0.53); p = 8.48E-04; power = 0.97; The overall rate of E. coli-ASP hypersensitivity was approximately four times higher for female patients with CC genotype compared to patients with at least one T allele (OR = 4.03 (1.48–10.94), p = 6.28E-03; power = 0.78), while no such association was found in male patients.On the other hand, in recessive model, patients with CC genotype of rs707176 had significantly higher risk to have ASP hypersensitivity until the end of the reinduction compared to patients harbouring at least one T allele (OR = 3.02 (1.36–6.73); p = 6.76E-03; power = 0.73; When we evaluated the genders separately in each risk category, it turned out that in the MR group the association of rs707176 with ASP HSR was confined only to females with a very high OR (OR = 11.56 (2.56–52.27); p = 1.48E-03; E. coli-ASP in the MR group was observed during the first dose of ASP in the reinduction  after a three-month-long break in ASP therapy. By the end of the E. coli-ASP treatment the cumulative incidence of E. coli-ASP hypersensitivity was 34% (84 out of 244) and 11% (6 out of 55) for patients with rs2055083 GG and AA/AG genotypes (p = 1.3E-3), respectively (E. coli-ASP hypersensitivity was 60% (12 out of 20) and 10% (3 out of 30) for patients with GG and AA/AG genotypes (p = 5.0E-4), respectively (E. coli-ASP) without any HSR to E. coli-ASP none of the patients developed E. coli-ASP hypersensitivity in further blocks.The highest incidence of HSRs to ectively . In caseectively . In ordeectively . After cE. coli-ASP hypersensitivity developed. A strong effect of rs2055083 could be detected on the minimum number of doses. The hazard ratio of the minor A allele in dominant model was 0.25 ; p = 1.8E-03). This means that the hazard rate of patients who have at least one A allele is approximately 75% less than that of the patients who are homozygous to the G allele. In contrast, the minor C allele of rs707176 in recessive model was proved to be a strong risk factor ; p = 0.0105). We also found a strong effect of rs4958351 on the minimum number of doses in the T-ALL subgroup on MR arm. The hazard rate of patients harbouring at least one A allele was about 91% less than patients with GG genotype ; p = 6.1E-04). Hence, the A allele of rs4958351 appeared to be a preventive factor in T-cell ALL.Furthermore, focusing on MR patients we used discrete-time survival analysis to assess the effect of the SNPs on the minimum number of doses at which E. coli-ASP hypersensitivity in various subgroups. Patients on HR treatment arm had significantly increased odds of developing E. coli-ASP hypersensitivity compared to those on SR and MR arm (OR = 5.88 (2.85–12.15); p = 2.00E-06 and OR = 10.50 (2.82–39.02); p = 4.49E-04, respectively). We found no differences in E. coli-ASP hypersensitivity rate between further subgroups  in the initial cohort of 322 paediatric patients, but these associations could not be detected in the smaller validation cohort of 163 children [Previously, Chen sitivity . Recentlsitivity . Chen etchildren .GRIA1 encodes a subunit (GluR1) of the ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor. These receptors are homomeric or heteromeric protein complexes consisting of GluR1-4 subunits, which are arranged to form a ligand-gated ion channel transmitting glutamatergic signals in the central nervous system (http://www.genecards.org/cgi-bin/carddisp.pl?gene=GRIA1).et al. described the presence of ionotropic glutamate receptors (iGluRs) on the surface of human T-lymphocytes. The same study has revealed the functionality of AMPA receptors by potentiation of Ca2+ influx upon T-cell activation [et al. demonstrated that during this interaction glutamate acts as a highly effective regulator in the initiation of T-cell mediated immune responses [ab ovo elevated level of plasma glutamate due to malignancy, the glutaminase activity of E. coli-ASP results in a significant rise of the cleavage product glutamate during treatment with a high interindividual variability [Recent studies have shown that glutamate acts also as an immunomodulator in addition to being a neurotransmitter ,16,23. Ftivation . Later, tivation –27. Moreiability . Taken tE. coli-ASP hypersensitivity in our total study cohort. However, we found that the effect of the A allele drastically differs on the susceptibility to ASP hypersensitivity in the different subtypes of ALL. While it is associated with significantly reduced risk to HSR in T-cell ALL (OR = 0.05), it is associated with a slightly although not significantly increased risk to HSR in pre-B-cell ALL. In contrast, Chen et al. and Rajić et al. have identified the A allele as a risk allele in the whole ALL cohort. However, perhaps because of the low number of T-ALL patients in their study cohorts, the different immunophenotypic subgroups have not been evaluated separately. To our knowledge, ours is the first study on the relation of rs4958351 to the risk of E. coli-ASP hypersensitivity in T-ALL as a separate subgroup; hence this opposite association in this subgroup of ALL patients could have been hidden up to this point.The rs4958351 SNP showed no association with GRIA1 gene appeared to be a strong preventive factor of E. coli-ASP hypersensitivity in a homogeneous cohort of MR ALL patients. No result has been published so far related to rs2055083. According to the results of the 1000 Genomes Project the rs2055083 is in linkage disequilibrium (LD) with another intronic SNP rs10515693  in the CEPH population. However, in the European (EUR) super population the regarding values were lower . Using the online Variant Effect Predictor (VEP) tool of Ensembl it has been revealed that the latter polymorphism is located in a regulatory region (ENSR00001294367), which is active in a human B-lymphocyte cell line GM12878 and acts as an enhancer . Based on this, one possible reason of association between rs2055083 and ASP allergy that rs2055083 is in LD with a regulatory variant in B-lymphocytes.In our study the intronic rs2055083 polymorphism in the GRIA1 gene, was associated with E. coli-ASP hypersensitivity in MR subgroup (p = 6.76E-03). The risk of CC homozygotes to HSR was very high in females (OR = 4.03 (1.48–10.94)), while no such effect could be observed in males which suggests a sex difference in the genetic background of ASP hypersensitivity.It has been shown in an Italian case-control association study that the allele distributions of rs707176 were different in DSM-IV-TR  schizophrenia controls and in cases . In our GRIA1 gene were intronic or synonymous variants. The functionality of these so-called silent polymorphisms is not yet clear. Intronic polymorphisms in the glutamate receptor subunit GluR2 have roles in directing RNA editing of the GluR2 coding sequence [GRIA1 has alternatively spliced transcript variants encoding different isoforms (http://www.genecards.org/cgi-bin/carddisp.pl?gene=GRIA1). Emerging data show that the fine balance of alternative splicing isoforms may also be altered by variants of exonic or intronic splicing regulatory elements [All the genetic variations we genotyped in sequence ,16,29. Felements . These fE. coli-ASP hypersensitivity by logistic regression between subgroups created by gender, age at diagnosis, risk category, immunophenotype of leukaemia and the dose of E. coli-ASP during induction on SR and MR arms. HR patients were at approximately six times higher odds of developing E. coli-ASP hypersensitivity compared to SR patients (p = 2.00E-06). The occurrence of E. coli-ASP hypersensitivity did not differ significantly between the SR and the MR groups. These findings can be explained by the considerably different treatment with more and greater doses of ASP as well as with many breaks in ASP therapy on HR arm compared to SR and MR arms.We tested the differences in the occurrence of E. coli-ASP hypersensitivity reactions was after a three-month-long break (consolidation phase) in ASP treatment during the first dose of reinduction on MR arm. It is in accordance with previously observed phenomena that reexposure of ASP after a hiatus increases the risk to HSRs [The highest occurrence of  to HSRs ,11. The  to HSRs ,31.E.coli-ASP was collected retrospectively from the files of the patients. This manner does not allow meticulous documentation and fine grading of hypersensitivity reactions.This study has several strengths and limitations. First, although the rate of relapsed patients of our cohort is similar to the relapse rate of the whole ALL population, the rate of died patients, however, is lower in our study population. Patients who died during the chemotherapy due to therapy resistant progressive disease or due to infections or toxicities of therapy are underrepresented in our ALL cohort. Furthermore, HSR data to E. coli-ASP. Fernandez et al. recently reported that the association between the rs4958351 variant and hypersensitivity was strongest among patients receiving native E. coli asparaginase compared to PEG-asparaginase [Our findings pertain to reactions to raginase .A small proportion of our DNA samples had been originated from the diagnostic clone of lymphoblasts. We hypothesized that the probability of the genetic alteration of the investigated polymorphisms was extremely low during leukaemogenesis. To verify this, we also performed the analysis excluding these samples and calculated the allele frequencies for the remission and for the diagnostic samples. Even in this way, the results remained statistically significant and we could not find substantial differences in the allele frequencies either (data not shown).GRIA1 gene could significantly influence the risk of ASP hypersensitivity. Furthermore, we found that the direction of the effect could be significantly different in the different subgroups of patients. In addition to previously published associations, we identified novel polymorphisms, which may serve to enlighten new details in the genetic background of E. coli-ASP hypersensitivity reactions. Replication in other cohorts is warranted to confirm our findings. Functional analysis of variants is needed, especially in case of the rs4958351 in T-ALL to elucidate its role in ASP allergy development.In conclusion, in our relatively large population we confirmed that genetic variations in the S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Extracellular matrix components such as hyaluronan (HA) and chondroitin sulfate (CS) were combined with a synthetic matrix of p(HEMA-co-METAC) (poly(2-hydroxyethylmethacrylate-co-2-methacryloxyethyltrimethylammonium)) at 1% and 2% w/w concentration following a previously developed procedure. The resulting semi-interpenetrating hydrogels were able to extensively swell in water incrementing their dry weight up to 13 fold depending on the glycosamminoglycan content and nature. When swollen in physiological solution, materials water uptake significantly decreased, and the differences in swelling capability became negligible. In physiological conditions, HA was released from the materials up to 38%w/w while CS was found almost fully retained. Materials were not cytotoxic and a biological evaluation, performed using 3T3 fibroblasts and an original time lapse videomicroscopy station, revealed their appropriateness for cell adhesion and proliferation. Slight differences observed in the morphology of adherent cells suggested a better performance of CS containing hydrogels.  A great number of naturally occurring polymers have been studied and proposed for the preparation of materials designed for Tissue Engineering applications .Specific biological features, such as bioresorbability and cytocompatibility, coupled to novel biotechnological processes for manufacturing, are at the basis of growing biomedical interest in these macromolecules. On the other hand, natural polymers are soluble or rapidly degraded in the physiological environment, and also exhibit poor mechanical properties. In order to overcome these drawbacks, they are often proposed in combination with other polymers  and/or in a chemically modified form (derivatized or crosslinked) ,7,8,9,10In a previous work we reported the synthesis and the characterization of a multiphase system in which a biopolymer, alginate , was combined with a synthetic poly(2-hydroxyethylmethacrylate-co-2-methacryloxyethyltrimethylammonium) (poly(HEMA-co-METAC)) matrix in a semi-interpenetrating structure . The resFrom a tissue engineering perspective, the presence of ECM molecules in a scaffold is fundamental for creating an environment mimicking the cellular surrounding ,6,7. As Actually, both HA and CS have been proposed as the main components of scaffolds for tissue regeneration. Chang and co-workers 2003) have developed a gelatin-CS-HA tri-copolymer scaffold, also proving its potentialities in cartilage tissue regeneration . Wang an have devBearing this literature background in mind, here we report the synthesis of semi-interpenetrating networks (semi-IPNs) in which HEMA and METAC were co-polymerized in the presence of aqueous solutions of HA or CS, and their characterization, in order to assess potential application in the biomedical field.Commercial 2-hydroxyethylmethacrylate (HEMA) and 2-methacryloxy ethyltrimethyl ammonium chloride (METAC) were purchased from Sigma-Aldrich Chemicals Co. . HEMA is known to contain residual EGDMA and a stabilizer, hydroquinonone monomethyl ether (0.001%), due to the fabrication process. Monomers were used without further purification. Hyaluronic acid sodium salt lot N. 0901142 was purchased from BioPhyl , Chondroitin sulfate sodium salt, extracted from shark fin, was a kind gift from IBSA . HA and CS disaccharide repeating units, along with structural formulas of HEMA and METAC monomers, are represented in Figure 1. TM), 3--2,5-diphenyltetrazolium bromide (MTT), Fetal Bovine Serum (FBS), penicillin, streptomycin and fungizone were all purchased from Life Technologies . Penicillin, streptomycin and fungizone were used at concentrations equal to 100 units/mL, 100 μg/mL, 2.5 μg/mL, respectively. 3T3 cells were purchased from ATCC .α-α’ azoisobutyronitrile (AIBN) was purchased from Fluka . Dulbecco’s Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagles Medium  in series that were preceded by a TSK-GEL guard column GMPWXL   were used. An isocratic elution with 0.1M NaNO3 aqueous solution (pH 7) at a flow rate of 0.6 mL/min was carried out. Analyses were performed at 40 °C with a running time of 1hr. Sample molecular weight , molecular size (hydrodynamic radius-Rh) and intrinsic viscosity ([η]) distributions were derived. The MHS curves (log [η] vs. log Mw) and, thus, the related constants were also directly obtained [The chromatographic analysis of the biopolymers was performed using the SEC-TDA (Size Exclusion Chromatography-Triple Detector Array) equipment by Viscotek . A detailed description of the system is reported elsewhere . Two TSKobtained .2O, p(HEMA-co-METAC)/HA1%, p(HEMA-co-METAC)/HA2%, p(HEMA-co-METAC)/CS1% and p(HEMA-co-METAC)/CS2%, were removed from the 3M transparency films and washed three times in de-ionized water for 24 hr to remove residual unreacted monomers. The rectangular polymeric membranes, in the swollen state, were cut to a circular shape to fit into a 12-well plate for chemico-physical and biological characterization and then dried in a forced-air circulation oven at 40 °C for 48–72 h. The hydrogels were synthesized following a previously described procedure . BrieflyThe water uptake for each material was studied in de-ionized water and in physiological solution . The swelling kinetic and the equilibrium swelling of the hydrogels were evaluated. sw = swollen sample weight; dw = dried initial sample weight. Experiments were run in triplicate.In all cases, the water uptake was determined by gravimetric measurements using an analytical balance . In particular, materials were immersed into the swelling solutions (200 mL aqueous medium/g of sample) and kept in a thermostatic bath at 37 °C. Specimens were removed at fixed intervals, 15–30 min up to 5 hr for kinetic studies, or after 24hr to assess the equilibrium swelling degree (equilibrium studies). Withdrawn samples were then blotted with filter paper to remove surface water and finally weighed. The swelling degree and the swelling ratio were calculated as follows:Biopolymer release from the polyelectrolyte matrices was determined through the following procedure: specimens of the dried materials (about 500 mg) were immersed in physiological solution (40 mL/g) and kept under stirring (200 rpm) at 37 °C for one week. At increasing time intervals, 1.5 mL of the medium were withdrawn and analyzed for the biopolymer content through the carbazole assay . The amo2, 95% air, humidified atmosphere and media were changed every 48 h.3T3 fibroblasts were routinely cultured in DMEM, supplemented with FBS (10% v/v), nonessential amino acids (1% v/v) and antibiotics. Cells were maintained at 37 °C in a 5% COin vitro cytotoxicity to assess the suitability of their use in biomedicine. The in vitro cytotoxicity was evaluated by means of the elution test method (ISO 10993-5), exposing 3T3 fibroblasts grown to near confluence to fluid extracts from the materials under investigation as accurately described elsewhere [The developed materials were tested for lsewhere , and thelsewhere . Cell vilsewhere .2O, p(HEMA-co-METAC)/CS1%, p(HEMA-co-METAC)/CS2%, p(HEMA-co-METAC)/HA1% and p(HEMA-co-METAC)/HA2% were treated twice with PBS containing antibiotics and then equilibrated overnight in DMEM at 37 °C in a 5% CO2 humidified atmosphere. Cells were then seeded on each disk (1–2 × 104 cells per cm2) and placed in a stage incubator of a time lapse videomicroscopy station in order to continuously monitor cell behavior in a 72 h experiments.Cytocompatibility was evaluated for all the materials obtained using 3T3 fibroblasts. In particular, p(HEMA-co-METAC)/H2), a thermostatic bath  and a motor control. The station is equipped with an innovative software  that permits a continuous on-line following of the experiment and the performing of images analysis. In particular, at about 3 hr after seeding, different areas on the hydrogel surfaces seeded with cells were selected. The software automatically drove the stage to return with micrometric precision on each selected X, Y, Z position at established time intervals, acquiring an image through a CCD gray-scale camera . Images relative to each area were automatically stored. Overall, the system was allowed to follow the movement of selected cells and, eventually, their adherence, spreading and proliferation on the material surface. The software was also used for image analysis in order to evaluate cell proliferation on each material. In particular, the adherent cells/cm2 were counted at time zero and every 12 h in 72 h experiments (proliferation curves were obtained by plotting cells number/cm2vs. time). The time lapse station  is composed by a inverted optical microscope , a motorized stage incubator  for each biopolymer are also reported in the HA and CS were analyzed by SEC-TDA to accomplish a complete hydrodynamic characterization. The report of the SEC-TDA analyses is presented in 2O. The samples evidenced rapid swelling and reached equilibrium within 4 hr. All hydrogels, as charged networks, presented high values of swelling degree (> 100%). As expected, the latter for the semi-interpenetrating hydrogels was considerably lower than that of the p(HEMA-co-METAC)/H2O, and decreased with the increase of the biopolymer content. Further, it is evident that, despite the same biopolymer concentration, the hydrogels containing CS showed a lower water uptake capacity with respect to the ones containing HA. In plateau. Results of the release studies are reported in The developed hydrogels were found not to be cytotoxic: viability of 3T3 fibroblasts  after 48hr of incubation in culture media conditioned with the tested hydrogels was at least 80% with respect to the control (data not shown). Micrographs of 3T3 fibroblasts on materials at 24, 48 and 72 h after seeding and the relative control (cells on TCP), as acquired by the time lapse station, are shown in 2vs. incubation time, normalized with respect to the number of seeded cells) for the novel materials compared to the control and to p(HEMA-co-METAC)/H2O are reported in −1vs. 0.027 h−1 of the control) while p(HEMA-co-METAC)/CS1% showed a minor growth-rate (proliferation-rate 0.022 h−1). The novel materials sustained proliferation better than the copolymer synthesized in the presence of water (growth-rate 0.022 h−1).Proliferation curves  matrix in a semi-interpenetrating structure gave materials  novel combinations have been exploited in the present work. In particular, p(HEMA-co-METAC) was combined with GAGs: specifically, HA and CS. Given that such polysaccharides are ECM components, the resulting materials were expected to be particularly suitable in mimicking cellular environment and predictably functional in specific applications ,5,6,7,8.4+ moieties.Swelling properties of the novel hydrogels were investigated in water and in physiological solution  since thAs expected, the water uptake of the hydrogels decreased when swollen in physiological solution due to the minor ion osmotic pressure (the difference in concentration of mobile ions between the gel and the solution is reduced when increasing the ionic strength of the swelling medium), and no significant differences among the diverse materials were recorded. This last result confirms our previous findings on p(HEMA-co-METAC)/alginate hydrogels , indicatAs shown in The absence of harmful extractables from the synthesized hydrogels was verified by indirect cytotoxicity tests performed according to ISO 10993-5 (data not shown). Further, in an attempt to propose synthesized materials for tissue engineering and to biologically compare them to the ones containing alginate, proliferation and morphology of cells on their surfaces have been investigated. As shown in It is worth mentioning that although HA and CS, as ECM components, are recognized as ideal scaffold materials, controversial literature exists on the effect of the polymers on cell adhesion. In particular, the polyanionic nature of HA was reported to thermodynamically not favor cellular adhesion ,30. SimiOverall, the combination of GAGs with a positively-charged synthetic matrix should allow an adhesive material to be obtained, due to the cationic surface, a mimicking cellular environment due to the GAG content and to contemporaneously tune physical properties by varying the nature and the content of the polysaccharide.Finally, it is worth underlying that, as expected, when using hyaluronan and chondroitin sulfate, the overall cellular response to the semi-IPNs improved with respect to the one found for p(HEMA-co-METAC)/alginate hydrogels . HA and CS were successfully introduced into p(HEMA-co-METAC) matrices, producing GAGs-based semi-interpenetrating hydrogels. The swelling properties of the developed materials were consistent with their polyelectrolyte nature and with the content and charge density of the biopolymer included. The outstanding water uptake and the partial release of the GAGs that was observed upon contact with aqueous medium potentially have advantageous effects in biomedical applications with respect to the previously synthesized p(HEMA-co-METAC)/alginate hydrogels. All developed materials proved biocompatible and suitable for cell adhesion and proliferation.


Vβ3 integrin that is expressed at high levels in the cell surface of TECs. To address this issue, we previously developed a cyclic RGD-equipped liposomal DDS (RGD-MEND) in which small interfering RNA (siRNA) was encapsulated. However, in the previous study, details of the preparation steps were not thoroughly examined. In this paper, to produce the most efficient delivery of therapeutic TECs, we explored optimum preparation conditions and components of the RGD-MEND. The cellular uptake and silencing ability of the RGD-MEND were investigated as a function of ligand density, poly(ethyleneglycol) linker length, and lipid composition. As a result, a knockdown efficiency that was five-fold higher than that of the previously reported one  was achieved.In recent years, anti-angiogenic therapy has attracted much interest because it is a versatile approach to treating most types of tumors, and therefore would be expected to be applicable for various cancers. Severe adverse events in patients treated with currently available anti-angiogenic therapeutics have, however, been reported, and these are caused by their inhibitory effects in normal tissue. To achieve an efficient anti-angiogenic therapy with minimal toxicity, a drug delivery system (DDS) specific to tumor endothelial cells (TECs) is needed. Cyclic RGD (cRGD) is a well-known ligand against α New ves3  and, the3 ,5. For tp-Toluidino)-6-naphthalenesulfonic acid (TNS) assay, which is a fluorescent molecule only in a lipophilic and cationic environment, revealed that the acid dissociation constant (pKa) of YSK05 was approximately 6.5 [Vβ3 integrin, which is specifically and highly expressed in TECs and some types of cancer cells [in vivo knockdown, very large amounts of siRNA (4.0 mg/kg) were needed to inhibit the TECs’ mRNA.To develop a new anti-angiogenic therapy without adverse effects, we attempted to combine a TEC-specific drug delivery system (DDS) and small interfering RNA (siRNA), which inhibits mRNA expression in a sequnce-specific manner. Recent studies revealed that mRNA expression in TECs is quite different from that of normal endothelial cells . Therefotely 6.5 . Furthertely 6.5 . We alsoer cells . Inhibit50). To accomplish this, we optimized the parameters for preparing the RGD-MEND to improve the efficiency of the in vivo knockdown in terms of ligand modification. We previously developed two types of MENDs that contained different lipid compositions for use in in vitro knockdown (MENDvitro) [in vivo hepatocyte knockdown (MENDvivo) [vivo was expected to show a higher gene knockdown in TECs than the MENDvitro, it was not possible to modify the MENDvivo with the RGD ligand under the same conditions that were used for the MENDvitro . Although we are not sure why ligand modification resulted in failure, the absence of a helper lipid in the composition of the MENDvivo might affect its insertion efficiency. Other groups reported that the addition of ethanol destabilized the lipid envelope [vivo.The aim of this study was to decrease the dose needed to inhibit the mRNA of TECs by half (EDNDvitro)  and in vENDvivo) ,11. The envelope  and the envelope . Based osn-grycero, poly(ethylene glycol)2000 (PEG-DMG), 1,2-distearoyl-sn-grycero, poly(ethylene glycol)2000 (PEG-DSG), 1,2-distearoyl-sn-grycerophosphoethanolamine, N-hydroxy-succinimide poly(ethylene glycol)2000, 3400 and 5000 , and 1-palmitoyl-2-oleoyl-sn-grycerophosphoethanol amine (POPE) were obtained from NOF CORPORATION . Griffonia simplicifolia isolectin B4 (GSIB4) was purchased from VECTOR Laboratories . Fluorescently hydrophobic molecules DiI and DiD were purchased from Life Technologies Corp. . Cyclic RGD (cyclo(Arg-Gly-Asn-d-Phe-Lys), cRGD) was obtained from Peptides International, Inc. . Dialysis membranes Spectra/Por 6 (MWCO 1000) were obtained from Spectrum Labs . All other chemicals used in the study were commercially available ones.YSK05 was synthesized as previously described . Cholest2k-RGD, PEG3.4k-RGD, PEG5k-RGD. The synthesis of these PEG-lipids were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry  dilution method as previously reported [t-BuOH at 7.5 mM, and siRNA was dissolved in 200 μL of 2 mM citric buffer (pH 4.0) in another tube. The siRNA solution was then gradually added to the lipid solution under vigorous mixing. The resulting mixture was then immediately diluted with 4.0 mL of PBS (−), and was then subjected to ultrafiltration with Vivaspin  twice to remove free siRNA molecules and t-BuOH from the preparation. If the MEND was fluorescently labeled, DiO, DiI, or DiD was added to the lipid solution before mixing with the siRNA solution at 0.5 mol% of total lipid. When the MENDs were modified with PEG-lipids, the RGD-MEND and specified amounts of PEG-lipids were incubated in 5 mM citric buffer (pH 5.0). Ethanol was added to the mixture under mixing to the indicated concentrations for 15–60 min at 30–60 °C. The MENDs were characterized by a dynamic light scattering method with a Zetasizer Nano ZS ZEN3600 instrument . The encapsulation efficiency (EE) and recovery rate (RR) of siRNA were determined using RNA quantification kit, RiboGreen (Life Technologies), as previously performed [The MEND was prepared by the tertiary butanol  when 70%–80% confluent.OS-RC-2 cells  and 4T1 cells (murine breast cancer) were obtained from the American Type Culture Collection. These cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% CO5 OS-RC-2 cells were plated onto six-well plates  24 h before the cellular uptake experiment. Fluorescent labeled MENDs were added to the wells at 24.4 μM (lipid), in which the lipid concentration was converted to approximately 100 nM of the siRNA concentration. The amount DiO or DiD-labeled MENDs added was adjusted to the sample showing the maximum FI by measuring the FI of each samples with Spectra MAX . After a 2-h incubation, cells were trypsinized, and the preparation then centrifuged . Supernatant was removed, and 500 μL of FACS buffer ) was added to the cell pellet. After the centrifugation , 500 μL of FACS buffer was added. This procedure was repeated twice to remove cell debris and unbound MENDs. Finally, the cell pellet was suspended in 1 mL of FACS buffer, and was used as a measurement sample. Samples were measured with FACSCalibur . Data were analyzed by Cell Quest (Becton Dickinson).To measure the ligand ability of the RGD-MENDs, 1.0 × 105 OS-RC-2 cells were plated on six-well plates  24 h before the experiment. Cells were transfected with indicated siRNA concentration of MEND for 24 h. Then, cells were lysed with 350 μL of TRIzol (Life Technologies Corp.), and then total RNA was extracted from cell lysates according to the manufacture’s protocol. For converting mRNA to cDNA, 1.0 μg of mRNA was reverse-transcribed with High Capacity RNA-to-cDNA kit (Life Technologies Corp.) as previously reported [To evaluate the knockdown efficacy of the MENDs, 1.0 × 10reported . The cDN6 cells. When tumor volume reached 100–200 mm3, the mice were used for in vivo experiments. These procedures were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the Guide for the Care and Use of Laboratory Animals (approved number: 13-0183 on 10 March 2015). For knockdown experiments, the MENDs were systemically injected into mice via the tail vein at the indicated dosages, and, after 24 h, tissue was excised and minced. TRIzol reagent (500 μL) was added to the minced tissue, and the tube of the TRIzol reagent was vigorously shaken by PreCellys . The following procedure for preparing cDNA was the same as that for the in vitro cultured cells.Balb/c mice  and ICR mice  were obtained from Japan SLC . Balb/c nude mice  were purchased from Japan CLEA . To prepare tumor-bearing mice, Balb/c (for 4T1) and Balb/c nude mice (for OS-RC-2) were anesthetized with diethylether, and the right flanks of the mice were inoculated with 1.0 × 10Ct method. All primers used in this manuscript were investigated in an amplification efficiency, and the sequences are listed in To determine the mRNA expression with qRT-PCR, 5.0 μL of 50-fold diluted cDNA was mixed with 0.075 μL of forward and reverse primes and 7.5 μL of THUNDIRBIRD qPCR Mix  and 2.35 μL of DDW . The mixture was then subjected to PCR with Light Cycler-480  according to the manufactures’ protocol. The expression level was calculated with ΔΔg, 5 min, 4 °C), and the fluorescence intensity of the homogenates was measured with a plate reader, SpectraMax Paradigm . Standard curve was prepared by adding a known quantity of the fluorescent labeled MEND to the homogenate from the non-treatment.Tumor accumulation was evaluated with fluorescent labeled MENDs. The DiI-labeled MEND was administered to tumor-bearing mice via the tail vein. Tumor tissue was collected 24 h after the administration, and the tumor tissues were homogenized in 50 mg tissue/mL 1% SDS by PreCellys. The homogenates were then centrifuged . These mice were injected with the DiD-labeled MENDs at a dose of 1.0 mg/kg. Forty microgram of FITC-labeled GSIB4 was injected via the tail vein 10 min before collecting the tumor tissue. The excised tumor tissue was cut into the half, and the specimens were observed using an A1 confocal imaging system .vivo. The lipid compositions of these two carriers are shown in Vβ3 integrin-positive cell line (v/v: ethanol (EtOH)/water) on facilitating the cellular uptake of MENDs by ligand modification. The cellular uptake amount was increased up to a 7.5 mol% modification  were regarded as a negative control. The extent of RGD-modification increased the cellular uptake of MENDs by three- to five-fold. Of these, the PEG5k-linker was generally inferior to the others in ligand ability. We next assessed the relationship between the gene silencing efficacy and ligand-PEG structure. Similarly, when the PEG-linker and ligand ratio was varied, the mRNA level of polo-like kinase 1 (PLK1) was determined by qRT-PCR (2k-DSPE and PEG2k-DSG), no gene silencing was observed. This weak silencing can be attributed to the inhibition of endosomal escape by PEGylation, in addition to a low cellular uptake of these carriers. Although the pre-formed MEND included PEG-DMG (C14-anchor), the PEG-lipid with the C14-anchor was rapidly removed from the surface of liposomes [We then evaluated the impact of the length of the PEG linker and the ligand density on cellular uptake and the gene knockdown efficacy of the RGD-MENDs. The amount of RGD-MENDs modified with 2000–5000 length and 1–5 mol% of RGD PEG-lipids that were internalized was measured by flow cytometry (FCM) A. Non-li qRT-PCR B. In theiposomes  and, henin vivo knockdown ability of the MENDvivo for optimization, murine breast cancer 4T1-bearing mice were used. After the MENDvivo that had been modified with various PEG-lipids was administered to mice via the tail vein, the target mRNA level was measured by qRT-PCR. In this experiment, Cd31 , which is specifically expressed in endothelial cells, was regarded as a target gene. Cd31 expression in tumor tissue was determined by qRT-PCR at 24 h after mice were treated with RGD-modified MENDs at a dose of 2.0 mg/kg , medium (137 d.nm), and large (282 d.nm) MENDs were administered to mice, the TEC mRNA expression level and the amount of tumor accumulation for the MENDs were measured. In gene silencing, all of the MENDs were similar, while the amount of tumor accumulation for the large MEND was higher than the others  value [To evaluate the .0 mg/kg A,B. The e others C. HoweveD) value . This muvivo (new one) with the RGD-modified MENDvitro  in in vivo gene silencing ability with human renal cell carcinoma OS-RC-2 cells. As shown in vivo or RGD-modified MENDvitro approximately a week after the inoculation . Accordingly, our carrier could be equal to the carrier that is now being clinically evaluated for gene silencing efficacy [We next compared the optimized RGD-modified MENDculation . In the .0 mg/kg . On the efficacy .vivo was observed by CLSM. Tumor tissue was observed 24 h after the injection of the fluorescent labeled MEND  in the liver, lung, kidney, and small intestine were determined (vivo (PEG-MEND) was regarded as a negative control. No gene silencing was observed in the lung, kidney, and small intestine (vivo three times in one day, one week after the final injection mRNA level of Vegfr2 was measured (Vegfr2 mRNA level of the continuously treated mice was the same as that for the non-treated mice. Accordingly, this inhibition would be transient and, therefore, the inhibition in liver endothelial cells by MENDs might not be a serious issue. Further study will be needed to circumvent this inhibition in off-target organs.To assess the possibility that unpredicted adverse effects could occur in off-target organs, the optimized RGD-modified MENDtermined . In the ntestine . The lunmeasured . The Veg50 was <0.75 mg/kg. From the view point of efficiency, the optimized RGD-MEND represents one of the most efficient siRNA carriers reported to date for targeting tumor endothelial cells.In this study, we established an efficient methodology for modifying a particle with a PEG-lipid. Based on this methodology, the conditions for PEG-lipid modification, the ligand density, and the length of the ligand were optimized. The optimized RGD-MEND showed an efficiency of 10-fold in gene silencing in tumor endothelial cells, and the ED